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In Vitro Propagation of Gastrochilus Matsuran (Makino) Schltr., an Endangered Epiphytic Orchid

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In Vitro Propagation of Gastrochilus Matsuran (Makino) Schltr., an Endangered Epiphytic Orchid plants Article In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid Hyeonjeong Kang 1, Kyung Won Kang 1, Doo Hwan Kim 2 and Iyyakkannu Sivanesan 2,* 1 Babo Orchid Farm, Namyangju-si, Gyeonggi-do 472-831, Korea; [email protected] (H.K.); [email protected] (K.W.K.) 2 Department of Bioresources and Food Science, Institute of Natural Science and Agriculture, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-450-0576 Received: 10 April 2020; Accepted: 17 April 2020; Published: 18 April 2020 Abstract: Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed 1 germination (93.3%) was achieved on 2 MS medium without vitamins plus 5% CW, 1 µM NAA, and 1 1.5 µM GA3. Secondary protocorm formation was best achieved on 2 MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid. Keywords: asymbiotic seed germination; protocorm; auxin; gibberellic acid; thidiazuron; coconut water; Orchidaceae 1. Introduction Gastrochilus matsuran (Makino) Schltr. (Orchidaceae), known as the purple-spotted gastrochilus, is a miniature epiphytic orchid native to Japan, Korea, and Taiwan. In Korea, G. matsuran is found on rocks and tree trunks in the low mountains of Gyeongsangnam-do and Jeju-do. It has a high economic value in the ornamental industry. The natural populations of G. matsuran are declining quickly because of overexploitation, climatic changes, and deforestation. Therefore, the epiphytic orchid has been designated as a rare and endangered species, and it is regionally protected by the law [1]. Natural propagation of orchids is frequently hindered by meager seed germination and slow growth [2]. Therefore, tissue culture is a viable alternative technique for large-scale multiplication and conservation of this endangered orchid. Although mass propagation of orchids has been achieved through adventitious shoot regeneration [3], multiple shoot induction [4], and somatic embryogenesis [5], extensive large-scale propagation through protocorm-like bodies, induced from various explants (including seeds), is often preferred by orchid researchers [6–9]. The in vitro asymbiotic seed germination technique has been effectively used for the conservation and feasible production of endangered orchids such as Cypripedium lentiginosum [10], Gastrochilus calceolaris [11], Gastrochilus japonicus [12], Pecteilis radiata [13], and Thrixspermum japonicum [14]. Plants 2020, 9, 524; doi:10.3390/plants9040524 www.mdpi.com/journal/plants Plants 2020, 9, 524 2 of 10 Several group of factors, such as seed age, culture medium composition, environmental conditions, Plants 2020, 9, x FOR PEER REVIEW 2 of 10 and genotype, influence the rate of asymbiotic in vitro seed germination [10]. The composition of the cultureSeveral media group is a of significant factors, such factor as that seed aff ectsage, asymbioticculture medium seed germination. composition, Mineralenvironmental nutrients, carbohydrates,conditions, and vitamins, genotype, amino influence acids, growththe rate hormonesof asymbiotic and organicin vitro acidsseed aregermination necessary [10]. for inThe vitro asymbioticcomposition embryo of the development culture media and is a protocormsignificant formationfactor that affects [2]. Activated asymbiotic charcoal seed germination. (AC), natural additives,Mineral andnutrients, plant growthcarbohydrates, regulators vitamins, (PGRs) amino are included acids, ingrowth the culture hormones media and to enhanceorganic acids orchid are seed germinationnecessary andfor in conversion vitro asymbiotic of seedlings embryo [2, 11development–14]. Successful and proceduresprotocorm formation for in vitro [2].regeneration Activated of taxonomicallycharcoal (AC), related natural species additives, such and as Gastrochilus plant growth calceolaris regulators[11 (PGRs)] and Gastrochilus are included japonicus in the culture[12] have beenmedia reported. to enhance However, orchid to seed the best germination of our knowledge, and conversion no information of seedlings is available[2,11–14].on Successful the in vitro propagationprocedures of forG. in matsuran vitro regeneration. In this study, of taxonomically we aimed to related develop species a procedure such as for Gastrochilus the micropropagation calceolaris [11] and Gastrochilus japonicus [12] have been reported. However, to the best of our knowledge, no of G. matsuran and reintroduction to its natural habitat. To establish a reliable and efficient procedure information is available o for mass propagation of G. matsuran and reduce the risk of its extinction, seeds from mature capsules (Figure1a) were used to study the e ffects of growth media, coconut water (CW), and PGRs on the asymbiotic germination, multiplication and conversion of protocorms, and plantlet development of G. matsuran in vitro. Here, we have described an efficient technique for the rapid propagation of G. matsuran. This in vitro propagation procedure would be helpful for mass production as well as conservation of this rare epiphytic orchid. FigureFigure 1. In1. vitroIn vitropropagation propagation of G. of matsuran G. matsuran:(a) Seed: (a) Seed capsule; capsule; (b) Induction (b) Induction of protocorm; of protocorm; (c) Induction (c) ofInduction secondary of protocorms; secondary protocorms; (d) Emergence (d) Emergence of leaf primordia; of leaf primordia; (e) Elongation (e) Elongation of leaf; of (f )leaf; Seedlings (f) development;Seedlings development; (g) Plantlets (g acclimatized) Plantlets acclimatized in the greenhouse; in the greenhouse; (h) Reintroduced (h) Reintroduced acclimatized acclimatized plantlets on plantlets on the tree trunk. Scale bar: (a-e) 0.1 mm; (f and g) 1 cm. the tree trunk. Scale bar: (a-e) 0.1 mm; (f and g) 1 cm. Plants 2020, 9, x FOR PEER REVIEW 3 of 10 2. Results 2.1. Impact of Culture Media on Seed Germination Microbial contamination is one of the serious problems limiting the successful extrapolation of plant tissue culture practices. The successful initiation of in vitro culture mostly depends on surface sterilization of explants because this is the primary source. Numerous surface microflora atttached to plant surfaces, grow faster than the cultured explants and release phytotoxic substances into the culture media, hindering positive outcomes. The surface sterilization procedure produced 98% sterile G. matsuran seeds. Seeds isolated from the mature capsules of G. matsuran were inoculated on various media containing 0.05% AC, 1% banana pulp, 0.2% peptone, 3% sucrose, and 0.8% plant agar for germination. Asymbiotic in vitro seed germination of G. matsuran was affected significantly (p ≤ 0.05) by the culture medium (Figure 2). Seed germination was observed within seven weeks of incubation. After 10 weeks of culture, pale green protocorms were produced (Figure 1b). Germination percentagesPlants 2020, 9, 524of 11.9%, 21.1%, 30.9%, 43.4%, and 25.8% were observed after 12 weeks of incubation3 ofon 10 Hyponex, Knudson C, Murashige and Skoog (MS) without vitamins, ½ MS without vitamins, and Vacin2. Results and Went media, respectively (Figure 2). Among the five nutrient media, best seed germination rate was achieved on ½ MS (without vitamins) medium. Therefore, ½ MS (without vitamins) medium was2.1. used Impact for of the Culture subsequent Media on seed Seed germination Germination experiments. Microbial contamination is one of the serious problems limiting the successful extrapolation of 2.2. Impact of CW and PGRs on Seed Germination plant tissue culture practices. The successful initiation of in vitro culture mostly depends on surface sterilizationThe asymbiotic of explants seed because germination this israte the of primary G. matsuran source. was Numerous affected significantly surface microflora (p ≤ 0.05) atttached by CW andto plant PGRs. surfaces, Supplementation grow faster of than CW theand cultured PGRs (indole-3-acetic explants and releaseacid (IAA), phytotoxic α-naphthaleneacetic substances into acid the (NAA),culture media,and gibberellic hindering acid positive (GA3) outcomes. in ½ MS The(without surface vitamins) sterilization medium procedure containing produced 0.05% 98% AC, sterile 1% bananaG. matsuran pulp,seeds. 0.2% Seedspeptone, isolated 3% fromsucrose, the matureand 0.8% capsules plant ofagarG. matsuran increasedwere the inoculated percentage on of various seed germinationmedia containing (Table 0.05%1). Among
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